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Abstract
Background
Toxins are key virulence determinants of pathogens and can impair the function of host immune cells, including platelets. Insights into pathogen toxin interference with platelets will be pivotal to improve treatment of patients with bacterial bloodstream infections.
Materials and Methods
In this study, we deciphered the effects of Staphylococcus aureus toxins α‐hemolysin, LukAB, LukDE, and LukSF on human platelets and compared the effects with the pore forming toxin pneumolysin of Streptococcus pneumoniae. Activation of platelets and loss of platelet function were investigated by flow cytometry, aggregometry, platelet viability, fluorescence microscopy, and intracellular calcium release. Thrombus formation was assessed in whole blood.
Results
α‐hemolysin (Hla) is known to be a pore‐forming toxin. Hla‐induced calcium influx initially activates platelets as indicated by CD62P and αIIbβ3 integrin activation, but also induces finally alterations in the phenotype of platelets. In contrast to Hla and pneumolysin, S. aureus bicomponent pore‐forming leukocidins LukAB, LukED, and LukSF do not bind to platelets and had no significant effect on platelet activation and viability. The presence of small amounts of Hla (0.2 µg/ml) in whole blood abrogates thrombus formation indicating that in systemic infections with S. aureus the stability of formed thrombi is impaired. Damage of platelets by Hla was not neutralized by intravenous immune globulins.
Conclusion
Our findings might be of clinical relevance for S. aureus induced endocarditis. Stabilizing the aortic‐valve thrombi by inhibiting Hla‐induced impairment of platelets might reduce the risk for septic (micro‐)embolization.
Complement resistance is an important virulence trait of Yersinia enterocolitica (Ye). The predominant virulence factor expressed by Ye is Yersinia adhesin A (YadA), which enables bacterial attachment to host cells and extracellular matrix and additionally allows the acquisition of soluble serum factors. The serum glycoprotein vitronectin (Vn) acts as an inhibitory regulator of the terminal complement complex by inhibiting the lytic pore formation. Here, we show YadA-mediated direct interaction of Ye with Vn and investigated the role of this Vn binding during mouse infection in vivo. Using different Yersinia strains, we identified a short stretch in the YadA head domain of Ye O:9 E40, similar to the ‘uptake region' of Y. pseudotuberculosis YPIII YadA, as crucial for efficient Vn binding. Using recombinant fragments of Vn, we found the C-terminal part of Vn, including heparin-binding domain 3, to be responsible for binding to YadA. Moreover, we found that Vn bound to the bacterial surface is still functionally active and thus inhibits C5b-9 formation. In a mouse infection model, we demonstrate that Vn reduces complement-mediated killing of Ye O:9 E40 and, thus, improved bacterial survival. Taken together, these findings show that YadA-mediated Vn binding influences Ye pathogenesis.
The iron-regulated surface determinant protein B (IsdB) of Staphylococcus aureus is involved in the acquisition of iron from hemoglobin. Moreover, IsdB elicits an adaptive immune response in mice and humans. Here, we show that IsdB also has impact on innate immunity. IsdB induces the release of proinflammatory cytokines, including IL-6 and IL-1β, in innate immune cells of humans and mice. In silico analysis and thermophoresis show that IsdB directly binds to TLR4 with high affinity. TLR4 sensing was essential for the IsdB-mediated production of IL-6, IL-1β, and other cytokines as it was abolished by blocking of TLR4-MyD88-IRAK1/4-NF-κB signaling. The release of IL-1β additionally required activation of the NLRP3 inflammasome. In human monocytes infected with live S. aureus, IsdB was necessary for maximal IL-1β release. Our studies identify S. aureus IsdB as a novel pathogen-associated molecular pattern that triggers innate immune defense mechanisms.
The Two-Component System 09 Regulates Pneumococcal Carbohydrate Metabolism and Capsule Expression
(2021)
Influenza A Virus (IAV), Staphylococcus aureus (staphylococci), and Streptococcus pneumoniae (pneumococci) are leading viral and bacterial causes of pneumonia. Dendritic cells (DCs) are present in the lower respiratory tract. They are characterized by low expression of co-stimulatory molecules, including CD80 and CD86 and high capacity of antigen uptake. Subsequently, DCs upregulate co-stimulatory signals and cytokine secretion to effectively induce T-cell priming. Here, we investigated these processes in response to bacterial and viral single as well as coinfections using human monocyte-derived (mo)DCs. Irrespective of single or coinfections, moDCs matured in response to IAV and/or staphylococcal infections, secreted a wide range of cytokines, and activated CD4+, CD8+ as well as double-negative T cells. In contrast, pneumococcal single and coinfections impaired moDC maturation, which was characterized by low expression of CD80 and CD86, downregulated expression of CD40, and a mild cytokine release resulting in abrogated CD4+ T-cell activation. These actions were attributed to the cholesterol-dependent cytotoxin pneumolysin (Ply). Infections with a ply-deficient mutant resulted in restored moDC maturation and exclusive CD4+ T-cell activation. These findings show that Ply has important immunomodulatory functions, supporting further investigations in specific modalities of Ply-DC interplay.
Epithelial cells are an important line of defense within the lung. Disruption of the epithelial barrier by pathogens enables the systemic dissemination of bacteria or viruses within the host leading to severe diseases with fatal outcomes. Thus, the lung epithelium can be damaged by seasonal and pandemic influenza A viruses. Influenza A virus infection induced dysregulation of the immune system is beneficial for the dissemination of bacteria to the lower respiratory tract, causing bacterial and viral co-infection. Host cells regulate protein homeostasis and the response to different perturbances, for instance provoked by infections, by post translational modification of proteins. Aside from protein phosphorylation, ubiquitination of proteins is an essential regulatory tool in virtually every cellular process such as protein homeostasis, host immune response, cell morphology, and in clearing of cytosolic pathogens. Here, we analyzed the proteome and ubiquitinome of A549 alveolar lung epithelial cells in response to infection by either Streptococcus pneumoniae D39Δcps or influenza A virus H1N1 as well as bacterial and viral co-infection. Pneumococcal infection induced alterations in the ubiquitination of proteins involved in the organization of the actin cytoskeleton and Rho GTPases, but had minor effects on the abundance of host proteins. H1N1 infection results in an anti-viral state of A549 cells. Finally, co-infection resembled the imprints of both infecting pathogens with a minor increase in the observed alterations in protein and ubiquitination abundance.
Bloodstream infections caused by Streptococcus pneumoniae induce strong inflammatory and procoagulant cellular responses and affect the endothelial barrier of the vascular system. Bacterial virulence determinants, such as the cytotoxic pore-forming pneumolysin, increase the endothelial barrier permeability by inducing cell apoptosis and cell damage. As life-threatening consequences, disseminated intravascular coagulation followed by consumption coagulopathy and low blood pressure is described. With the aim to decipher the role of pneumolysin in endothelial damage and leakage of the vascular barrier in more detail, we established a chamber-separation cell migration assay (CSMA) used to illustrate endothelial wound healing upon bacterial infections. We used chambered inlets for cell cultivation, which, after removal, provide a cell-free area of 500 μm in diameter as a defined gap in primary endothelial cell layers. During the process of wound healing, the size of the cell-free area is decreasing due to cell migration and proliferation, which we quantitatively determined by microscopic live cell monitoring. In addition, differential immunofluorescence staining combined with confocal microscopy was used to morphologically characterize the effect of bacterial attachment on cell migration and the velocity of gap closure. In all assays, the presence of wild-type pneumococci significantly inhibited endothelial gap closure. Remarkably, even in the presence of pneumolysin-deficient pneumococci, cell migration was significantly retarded. Moreover, the inhibitory effect of pneumococci on the proportion of cell proliferation versus cell migration within the process of endothelial gap closure was assessed by implementation of a fluorescence-conjugated nucleoside analogon. We further combined the endothelial CSMA with a microfluidic pump system, which for the first time enabled the microscopic visualization and monitoring of endothelial gap closure in the presence of circulating bacteria at defined vascular shear stress values for up to 48 h. In accordance with our CSMA results under static conditions, the gap remained cell free in the presence of circulating pneumococci in flow. Hence, our combined endothelial cultivation technique represents a complex in vitro system, which mimics the vascular physiology as close as possible by providing essential parameters of the blood flow to gain new insights into the effect of pneumococcal infection on endothelial barrier integrity in flow.
The M protein of Streptococcus canis (SCM) is a virulence factor and serves as a surface-associated receptor with a particular affinity for mini-plasminogen, a cleavage product of the broad-spectrum serine protease plasmin. Here, we report that SCM has an additional high-affinity immunoglobulin G (IgG) binding activity. The ability of a particular S. canis isolate to bind to IgG significantly correlates with a scm-positive phenotype, suggesting a dominant role of SCM as an IgG receptor. Subsequent heterologous expression of SCM in non-IgG binding S. gordonii and Western Blot analysis with purified recombinant SCM proteins confirmed its IgG receptor function. As expected for a zoonotic agent, the SCM-IgG interaction is species-unspecific, with a particular affinity of SCM for IgGs derived from human, cats, dogs, horses, mice, and rabbits, but not from cows and goats. Similar to other streptococcal IgG-binding proteins, the interaction between SCM and IgG occurs via the conserved Fc domain and is, therefore, non-opsonic. Interestingly, the interaction between SCM and IgG-Fc on the bacterial surface specifically prevents opsonization by C1q, which might constitute another anti-phagocytic mechanism of SCM. Extensive binding analyses with a variety of different truncated SCM fragments defined a region of 52 amino acids located in the central part of the mature SCM protein which is important for IgG binding. This binding region is highly conserved among SCM proteins derived from different S. canis isolates but differs significantly from IgG-Fc receptors of S. pyogenes and S. dysgalactiae sub. equisimilis, respectively. In summary, we present an additional role of SCM in the pathogen-host interaction of S. canis. The detailed analysis of the SCM-IgG interaction should contribute to a better understanding of the complex roles of M proteins in streptococcal pathogenesis.
Like eukaryotes, different bacterial species express one or more Ser/Thr kinases and phosphatases that operate in various signaling networks by catalyzing phosphorylation and dephosphorylation of proteins that can immediately regulate biochemical pathways by altering protein function. The human pathogen Streptococcus pneumoniae encodes a single Ser/Thr kinase-phosphatase couple known as StkP-PhpP, which has shown to be crucial in the regulation of cell wall synthesis and cell division. In this study, we applied proteomics to further understand the physiological role of pneumococcal PhpP and StkP with an emphasis on phosphorylation events on Ser and Thr residues. Therefore, the proteome of the non-encapsulated D39 strain (WT), a kinase (ΔstkP), and phosphatase mutant (ΔphpP) were compared in a mass spectrometry based label-free quantification experiment. Results show that a loss of function of PhpP causes an increased abundance of proteins in the phosphate uptake system Pst. Quantitative proteomic data demonstrated an effect of StkP and PhpP on the two-component systems ComDE, LiaRS, CiaRH, and VicRK. To obtain further information on the function, targets and target sites of PhpP and StkP we combined the advantages of phosphopeptide enrichment using titanium dioxide and spectral library based data evaluation for sensitive detection of changes in the phosphoproteome of the wild type and the mutant strains. According to the role of StkP in cell division we identified several proteins involved in cell wall synthesis and cell division that are apparently phosphorylated by StkP. Unlike StkP, the physiological function of the co-expressed PhpP is poorly understood. For the first time we were able to provide a list of previously unknown putative targets of PhpP. Under these new putative targets of PhpP are, among others, five proteins with direct involvement in cell division (DivIVA, GpsB) and peptidoglycan biosynthesis (MltG, MreC, MacP).